Hybrid Hepatocyte Growth Factor Gene Having High Expression Efficiency of Two Heterotypes of Hepatocyte Growth Factor

ABSTRACT

The present invention relates to a hybrid Hepatocyte Growth Factor (HGF) gene which is prepared by inserting an inherent or foreign intron between exons 4 and 5 in HGF cDNA, which has a base sequence of SEQ ID NO: 2. The gene has high expression efficiency and simultaneously expresses two heterotypes of HGF and dHGF (deleted variant HGF). Further the gene may be used for treating or preventing ischemic or liver diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/957,170, filed Dec. 14, 2007, now allowed, which is a divisional ofU.S. application Ser. No. 10/944,277, filed Sep. 20, 2004, now issuedU.S. Pat. No. 7,812,146, which is a continuation of InternationalApplication No. PCT/KR03/00548, filed Mar. 20, 2003, each of which isherein incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:sequence listing.ascii.txt, Size: 29,896 bytes; Date of Creation: Oct.12, 2010) filed with the application is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a highly efficient hybrid HepatocyteGrowth Factor (HGF) gene which simultaneously expresses two heterotypesof HGF.

2. Related Art

The present invention relates to a hybrid HGF gene prepared by insertingan inherent or foreign intron between exons 4 and 5 in HGF cDNA, whichhas higher expression efficiency than HGF cDNA and simultaneouslyexpresses two heterotypes of HGF and dHGF (deleted variant HGF).

HGF is a heparin binding glycoprotein called a scatter factor. A geneencoding HGF is located at chromosome 7q21.1 and comprises 18 exons and17 introns, having the nucleotide sequence of SEQ ID NO: 1 (Seki T., etal., Gene 102:213-219 (1991)). A transcript of about 6 kb is transcribedfrom the HGF gene, and then, a polypeptide HGF precursor consisting of728 amino acids is synthesized therefrom. Simultaneously, a polypeptideof dHGF precursor consisting of 723 amino acids is also synthesized byan alternative splicing of the HGF gene. The biologically inactiveprecursors may be converted into active forms of disulfide-linkedheterodimer by protease in serum. In the heterodimers, the alpha chainhaving a high molecular weight forms four kringle domains and anN-terminal hairpin loop like a preactivated peptide region ofplasminogen. The kringle domains of a triple disulfide-bonded loopstructure consisting of about 80 amino acids may play an important rolein protein-protein interaction. The low molecular weight beta chainforms an inactive serine protease-like domain. dHGF consisting 723 aminoacids is a polypeptide with deletion of five amino acids in the 1stkringle domain of the alpha chain, i.e., F, L, P, S and S.

It has been recently reported that both of HGF and dHGF have severalbiological functions, e.g., promoting the growth and morphogenesis ofepithelial cell, melanocyte and endothelial cell. However, they aredifferent in terms of immunological or biological properties.

For example, HGF shows about 20-fold, 10-fold and 2-fold higheractivities than dHGF in promoting DNA synthesis in human umbilical cordvenous endothelial cell, arterial smooth muscle cell and NSF-60 (murinemyeloblast cell), respectively. dHGF shows about 3-fold and 2-foldhigher activities than HGF in promoting DNA synthesis of LLC-PK1 (pigkidney epithelial cell), and OK (American opossum kidney epithelialcell) and mouse interstitial cell, respectively. HGF has a 70-foldhigher solubility in PBS than dHGF. Several anti-dHGF monoclonalantibodies recognize only dHGF, but not HGF or a reduced form of dHGF,which implies structures of HGF and dHGF are different. Accordingly, thesimultaneous synthesis of HGF and dHGF in vivo suggests that theybiologically interact with each other (Shima, N. et al., Biochemical andBiophysical Research Communications 200:808-815 (1994)).

HGF secreted from mesoderm-derived cells has various biologicalfunctions, e.g., 1) inducing epithelial cells into a tubular structure;2) stimulating vascularization from endothelial cells in vitro and invivo; 3) regeneration of liver and kidney, owing to its anti-apoptosisactivity; 4) organogenesis of kidney, ovary and testis; 5) controllingosteogenesis; 6) stimulating the growth and differentiation of erythroidhematopoietic precursor cells; and 7) axon sprouting of neurons (Stella,M. C. and Comoglio, P.M., The International Journal of Biochemistry &Cell Biology 31:1357-1362 (1999)). Based on these various functions, HGFor a gene encoding HGF may be developed as a therapeutic agent fortreating ischemic or liver diseases. Actually, in vivo, the HGF mayexist as either HGF or dHGF, and therefore, the coexpression of HGF anddHGF is important for maximizing the therapeutic effect. Accordingly,the present inventors have endeavored to develop a hybrid HGF gene whichcan simultaneously express HGF and dHGF with a high efficiency for genetherapy.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the present invention to providea hybrid HGF gene which simultaneously expresses two heterotypes of HGF.

In accordance with one aspect of the present invention, there is providethe hybrid HGF gene having an inherent or foreign intron is insertedbetween exons 4 and 5 of HGF cDNA.

It is another object of the present invention to provide a recombinantvector comprising the hybrid HGF gene and a microorganism transformedwith the above vector.

It is a still further object of the present invention to provide apharmaceutical composition for treating or preventing ischemic or liverdiseases, which comprises the HGF gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings which respectivelyshow:

FIG. 1: a schematic diagram of HGF-X prototype illustrating thepositions of the gene fragments.

FIG. 2: a process for cloning gene fragments from HepG2 genomic DNA.

FIG. 3: a process for cloning gene fragments from human placenta cDNA.

FIGS. 4A and 4B: processes for preparing expression vectors pCK-HGF-X.

FIG. 5: a process for preparing expression vectors pCK-cHGF andpCK-dHGF.

FIG. 6: a process for preparing expression vectors pCP-HGF-X family.

FIG. 7: a process for preparing expression vectors pCP-cHGF andpCP-dHGF.

FIG. 8: gene expression levels of pCP-cHGF, pCP-dHGF and pCP-HGF-X.

FIG. 9: gene expression patterns of pCP-cHGF, pCP-dHGF and pCP-HGF-Xobserved by electrophoresis on 12% polyacrylamide gel.

FIG. 10: gene expression levels of pCP-cHGF, pCP-dHGF and pCP-HGF-X7, invivo.

FIG. 11: angiographic vessel count of two groups of rabbits which weresubject to administrating pCP and pCP-HGF-X7, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The hybrid Hepatocyte Growth Factor (HGF) gene of the present inventioncomprises cDNA corresponding to the exons 1 to 18, and an inherent orforeign intron inserted between exons 4 and 5 of the cDNA. The introncomprises a fragment of the inherent intro or a recombinant sequence.

An embodiment of the hybrid HGF gene of the present invention comprisingthe inherent intron is 7113 bp long and has the nucleotide sequence ofSEQ ID NO: 2. The hybrid HGF gene simultaneously expresses both HGF anddHGF, and has higher expression efficiency than HGF cDNA.

Codon degeneracy enables the hybrid HGF gene of the present invention tobe modified or changed in the coding and/or non-coding region withoutaltering the amino acid sequence of the protein and the expression ofthe gene. Accordingly, polynucleotides which is substantially identicalto the hybrid HGF gene of SEQ ID NO:2, and the fragments thereof fallwithin the scope of the invention. “Substantially identical” means thatthe sequence homology is not less than 80%, preferably not less than90%, and more preferably not less than 95%.

A hybrid HGF gene may comprise a fragment of inherent intron optionallyhaving a small recombinant sequence inserted thereinto between exons 4and 5 of HGF cDNA. Herein, such a hybrid HGF gene comprising a fragmentof inherent intron designates “HGF-X”. HGFX-6, HGF-X7 and HGF-X8 havingthe nucleotide sequence of SEQ ID Nos: 19 to 21, respectively, arepreferred.

The hybrid HGF gene of the present invention is synthesized and insertedinto an expression vector, according to the known genetic engineeringmethods. Then, the vector can be introduced into an appropriate hostcells such as E. coli and yeast. For example, Escherichia coli Top10F′may be transfected with HGF-X7 gene of the present invention.Escherichia coli Top10F′ pCK-HGFX7 and Escherichia coli Top10F′pCP-HGFX7 then obtained were deposited as the accession numbersKCCM-10361 and KCCM-10362, respectively, on Mar. 12, 2002.

By using the transformed cells, the gene of the present invention andthe protein encoded thereby may be produced on a large scale.

The vector of the present invention may selectively comprise sequence(s)for regulating gene expression such as promoter or terminator,self-replication sequence and secretory signal, depending on host cells.

Further, the present invention comprises a pharmaceutical compositionfor treating or preventing ischemic and liver diseases, which comprisesthe hybrid HGF gene or the vector comprising the gene as an activeingredient. Preferably, the composition is formulated for injection.

The composition of the present invention may further comprisepharmaceutically acceptable carriers. Any of the conventional proceduresin the pharmaceutical field may be used to prepare oral formulationssuch as tablets, capsules, pills, granules, suspensions and solutions;rejection formulations such as solutions, suspensions, or dried powdersthat may be mixed with distilled water before injection;locally-applicable formulations such as ointments, creams and lotions;and other formulations.

Carriers generally used in the pharmaceutical field may be employed inthe composition of the present invention. For example,orally-administered formulations may include binders, emulsifiers,disintegrating agents, excipients, solubilizing agents, dispersingagents, stabilizing agents, suspending agents, coloring agents orspicery. Injection formulations may comprise preservatives, unagonizingagents, solubilizing agents or stabilizing agents. Preparation for localadministration may contain bases, excipients, lubricants orpreservatives. Any of the suitable formulations known in the art(Remington's Pharmaceutical Science [the new edition], Mack PublishingCompany, Eaton Pa.) may be used in the present invention.

The inventive composition can be clinically administered as various oraland parenteral formulations. A suitable formulation may be preparedusing such excipients as additives, enhancers, binders, wetting agents,disintegrating agents and surfactants, or diluents. Solid formulationsfor oral administration include pills, tablets, dusting powder, granulesand capsules. Those solid formulations may be prepared by mixing one ormore excipients, e.g. starch, calcium carbonate, sucrose, lactose andgelatin with dibenzylbuthyllacton lignan derivatives. Also, lubricantssuch as magnesium stearate and talc may be included in the presentformulation. Liquid formulations for oral administration includesuspension, solution, emulsion and syrup. Those formulations may containwetting agents, sweeteners, aromatics and preservatives, in addition togeneral simple diluents such as water and liquid paraffin. Formulationsfor parenteral administration include sterilized aqueous solution,suspension, emulsion, freeze-dried alternative treatment andsuppositories. Water-insoluble excipients and suspending agents comprisevegetable fats such as propylene glycol, polyethylene glycol and oliveoil, and injectable esters such as ethyl oleate. Witepsol®, Macrogol®,Tween® 61, cacao fats, laurin fats and glycerogelatins may be used asbases of suppositories.

The inventive composition may be administered orally or via parenteralroutes such as intravenous, intramuscular, subcutaneous, intraabdominal,sternal and arterial injection or infusion, or topically through rectal,intranasal, inhalational or intraocular administration.

It should be understood that the typical daily dose of composition ofthe present invention ought to be determined in light of variousrelevant factors including the conditions to be treated, the chosenroute of administration, the age, sex and body weight of the individualpatient, and the severity of the patient's symptom, and can beadministrated in a single dose or in divided dose. Therefore, the dailydose should not be construed as a limitation to the scope of theinvention in any way.

The following Examples are given for the purpose of illustration only,and are not intended to limit the scope of the invention.

Example 1 Preparation of Hybrid Gene Constructs Encoding Human HGF

(1) Cloning of HGF Gene Fragments Obtained from Genomic DNA

Human HepG2 cells (ATCC Accession NO: HB-8065) were suspended in TESbuffer (10 mM Tris-HCl; 1 mM EDTA; 0.7% SDS) and treated with 400 μg/Mlof proteinase K at 50° C. for 1 hour. Subsequently, genomic DNA wasextracted from the cell suspension by phenol/chloroform extraction andethanol precipitation according to the conventional method in the art.

In the PCR amplification, the extracted genomic DNA was employed as atemplate DNA. As primer pairs, the synthetic nucleotides of SEQ ID NOs:3 and 4 were employed to obtain DNA fragments containing: HGF genefragment 2 (HGF-F2), SEQ ID NOs: 3 and 5; HGF-F3, SEQ ID NOs: 6 and 7;HGF-F5, SEQ ID NOs: 8 and 7; HGF-F7, SEQ ID NOs: 9 and 7; HGF-F8, SEQ IDNOs: 10 and 7; HGF-F6, respectively (FIG. 1). The PCR amplificationmixture was prepared by mixing 1 μl of template DNA, 1 μl each of primer(10 pmol/μl), 10 μl of dNTP (10 mM), 3.5 unit of Expand High Fidelityenzyme (Gibco BRL, USA) and 10 μl of enzyme buffer solution and adjustedto a final volume of 100 μl with distilled water. 30 cycles of the PCRamplification was carried out, each cycle consisting of 1 min at 94° C.,1 min at 55° C. and 30 sec at 72° C. The primers used herein and theamplified gene fragments obtained therefrom are shown in Table 1.

TABLE 1 5′ primer 3′ primer Amplified fragment gHGF3 (SEQ ID NO: 3) gHGF4 (SEQ ID NO: 4) HGF gene fragment 2 (HGF-F2) gHGF3 (SEQ ID NO: 3)gHGF10 (SEQ ID NO: 5) HGF gene fragment 3 (HGF-F3) gHGF5 (SEQ ID NO: 6)gHGF7 (SEQ ID NO: 7) HGF gene fragment 5 (HGF-F5) gHGF12 (SEQ ID NO: 8)gHGF7 (SEQ ID NO: 7) HGF gene fragment 7 (HGF-F7) gHGF13 (SEQ ID NO: 9)gHGF7 (SEQ ID NO: 7) HGF gene fragment 8 (HGF-F8) gHGF6 (SEQ ID NO: 10)gHGF7 (SEQ ID NO: 7) HGF gene fragment 6 (HGF-F6)

The amplified HGF-F2 comprised the sequence ranging from 392 to 2247 ofhuman HGF cDNA prototype (HGF-X1; composed of exons 1 to 4-intron4-exons 5 to 18) of SEQ ID NO: 2; HGF-F3, the sequence ranging from 392to 727; HGF-5, the sequence ranging from 2229 to 5471; HGF-F6, thesequence ranging from 5117 to 5471; HGF-F7, the sequence ranging from3168 to 5471; and HGF-F8, the sequence ranging from 4168 to 5471.

The amplified HGF gene fragments were each inserted into the multiplecloning site of pGEM-T easy vector (Promega, WI, USA) to obtain pGEM-Teasy-HGF-F2, pGEM-T easy-HGF-F3, pGEM-T easy-HGF-F5, pGEM-T easy-HGF-F6,pGEM-T easy-HGF-F7 and pGEM-T easy-HGF-F8, respectively (FIG. 2). Thenucleotide sequences of the amplified HGF gene fragments were confirmedby a sequence analysis.

(2) Cloning of HGF Gene Fragments Obtained from cDNA

In the PCR amplification, human placenta cDNA (Clontech, CA, USA) wasemployed as a template DNA under the same condition as described inExample 1. As primer pairs, the synthetic oligonucleotides of SEQ IDNOs: 11 and 12, and SEQ ID NOs: 13 and 14 were employed to obtain DNAfragments containing HGF-F1 and HGF-F4, respectively. Further, DNAfragments containing cDNAs of HGF gene (cHGF) and deleted HGF gene(dHGF) were amplified by PCR using synthetic oligonucleotides of SEQ IDNOs: 15 and 16 as a primer pair, respectively. dHGF is a HGF gene withdeletion of 5 base sequences.

The primers used herein and the amplified gene fragments obtainedtherefrom are shown in Table 2.

TABLE 2 5′ primer 3′ primer Amplified fragment gHGF1 (SEQ ID NO: 11)gHGF2 (SEQ ID NO: 12) HGF gene fragment 1 (HGF-F1) gHGF8 (SEQ ID NO: 13)gHGF9 (SEQ ID NO: 14) HGF gene fragment 4 (HGF-F4) cHGF5 (SEQ ID NO: 15)cHGF3 (SEQ ID NO: 16) HGF gene cDNA (cHGF) dHGF gene cDNA (dHGF)

The amplified HGF-F1 and HGF-F4 comprised the nucleotide sequencesranging from 1 to 402 and from 6533 to 7113 of SEQ ID NO: 2 of human HGFcDNA prototype, respectively. HGF gene cDNA comprised the nucleotidesequence ranging from 1 to 2184 of SEQ ID NO: 1 of human HGF gene, anddHGF gene cDNA has the same sequence as HGF gene cDNA except for thedeletion of the sequence ranging from 483 to 495.

The amplified fragments of HGF gene were each inserted into the multiplecloning site of pGEM-T easy vector (Promega, WI, USA) to obtain pGEM-Teasy-HGF-F1, pGEM-T easy-HGF-F4, pGEM-T easy-cHGF and pGEM-T easy-dHGF,respectively (FIG. 3). The nucleotide sequences of the human HGF genefragments, HGF gene cDNA and dHGF gene cDNA were confirmed by sequenceanalyses.

(3) Preparation of Hybrid HGF Gene Constructs

Hybrid HGF gene constructs of genomic DNA and cDNA were prepared bycombining the fragments of HGF gene cloned in steps (1) and (2) asfollows (FIGS. 4A and 4B).

Plasmid pGEM-T-easy-HGF-F1 was treated with HindIII/BamHI to obtainHGF-F1. Plasmid pCK (see PCT International Publication NO: WO/0040737)was treated with HindIII/BamHI, and HGF-F1 was inserted thereinto toobtain pCK-F1. And then, plasmids pGEM-T-easy-HGF-F2 andpGEM-T-easy-HGF-F3 were treated with MluI/BamHI to obtain HGF-F2 andHGF-F3, respectively. pCK-1 was treated with MluI/BamHI, and then HGF-F2and HGF-F3 were inserted thereinto to obtain pCK-F12M and pCKF13M. TheMluI restriction site of vectors pCK-F12M and pCK-F13M was substitutedwith an HgaI restriction site by employing a site-directed mutagenesiskit (Stratagene, CA, USA) to obtain pCK-F12 and pCK-F13, respectively.

Further, plasmid pGEM-T-easy-HGF-F4 was treated with BamHI/XbaI toobtain HGF-F4. pCK-F12 and pCK-F13 were treated with BamHI/XbaI, andHGF-F4 was inserted thereinto to obtain pCK-F124 and pCK-F134,respectively. And then, plasmids pGEM-T-easy-HGF-F5, pGEM-T-easy-HGF-F6,pGEM-T-easy-HGF-F7 and pGEM-T-easy-HGF-F8 were treated with BamHI/XhoIto obtain HGF-F5, HGF-F6, HGF-F7 and HGF-F8, respectively. pCK-F124 andpCK-F134 were treated with BamHI/XhoI, and then HGF-F5, HGF-F6, HGF-F7and HGF-F8 were inserted thereinto to obtain pCK-F1254 and pCK-F1264,pCK-F1274, pCK-F1284, pCK-F1354, pCK-F1364, pCK-F1374 and pCK-F1384,respectively.

And then, pGEM-T easy-cHGF was treated with XhoI to obtain cDNA fragmentof HGF gene (HGF-XhoI) of about 1100 bp. Then, HGF-XhoI was insertedinto pCK-F1254, pCK-F1264, pCK-F1274, pCK-F1284, pCK-F1354, pCK-F1364,pCK-F1374 and pCK-F1384 to obtain pCK-HGF-X1, pCK-HGF-X2, pCK-HGF-X3,pCK-HGF-X4, pCK-HGF-X5, pCK-HGF-X6, pCK-HGF-X7 and pCK-HGF-X8,respectively. On the other hand, pGEM-T easy-cHGF and pGEM-T easy-dHGFwere treated with BamHI to obtain HGF gene cDNA and dHGF gene cDNA.Then, HGF gene cDNA and dHGF gene cDNA were inserted into the BamHIrestriction site of pCK to obtain pCK-cHGF and pCK-dHGF, respectively(FIG. 5).

(4) Preparation of an Expression Vector Containing a Hybrid HGF GeneConstruct

Plasmid pcDNA3.1 (Invitrogen, USA) was digested with NdeI, treated withthe Klenow fragment to build blunt ends, and then digested with NheI toobtain a DNA fragment containing human cytomegalovirus promoter.Plasmids pCK-HGF-X1, pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X4, pCK-HGF-X5,pCK-HGF-X6, pCK-HGF-X7 and pCK-HGF-X8 were digested with SnaBI, treatedwith the Klenow fragment to make blunt ends and digested with NheI, andthen the above DNA fragment containing human cytomegalovirus promoterwas inserted thereinto to obtain pCP-HGF-X1, pCP-HGF-X2, pCP-HGF-X3,pCP-HGF-X4, pCP-HGF-X5, pCP-HGF-X6, pCP-HGF-X7 and pCP-HGF-X8,respectively (FIG. 6).

Plasmid pcDNA3.1 (Invitrogen, USA) was digested with NheI, treated withthe Klenow fragment to make blunt ends and digested with NdeI to obtainthe DNA fragment containing human cytomegalovirus promoter. pCK-cHGF andpCK-dHGF were digested with MluI, treated with the Klenow fragment tomake blunt ends and digested with NdeI, and then the above DNA fragmentcontaining human cytomegalovirus promoter was inserted thereinto toobtain pCP-cHGF and pCP-dHGF, respectively (FIG. 7).

Example 2 Examination of the Expression Efficiency of Hybrid HGF GeneConstruct and the Coexpression of HGF/dHGF

Studies were conducted to examine whether the hybrid HGF gene constructs(HGF-X1 to HGF-X8) obtained in Example 1 can simultaneously express HGFand dHGF and whether there is any difference in the gene expressionlevel between hybrid HGF gene constructs and HGF cDNA.

(1) Gene Expression Efficiency

First, 5 μg of pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7 andpCP-HGF-X8 were transfected into 5×10⁶ cells of 293 cell (ATCC CRL 1573)together with 0.5 μg of DONAI-LacZ (TAKARA SHUZO, Japan) DNA usingFuGENE6 (Gibco BRL, MD, USA), according to the manufacturer'sinstructions. At this time, 5 μg each of pCP-cHGF and pCP-dHGF were usedas controls, and DONAI-LacZ DNA was used to calibrate the infectionefficiency. 3 hours after transfection, cells were re-fed with a freshmedium and further cultured for 48 hours. The culture solution thusobtained was divided into two parts. One part of the 293 cell culturesolution was subjected to RNA extraction, and the other, to measurementof LacZ activity. The LacZ activity was measured using an activitymeasuring kit (Stratagene, CA, USA) according to the manufacturer'sinstructions.

In order to compare the gene expression levels, the amount of HGF in thecell culture was measured by an enzyme-linked immunosorbent assay kit(ELISA, R&D System, MN, USA). After calibrating the infection efficiencyby the measured LacZ activity, the expression level of HGF-X gene wasfound to be from 20 to 150-fold higher than those of HGF cDNA and dHGFcDNA (FIG. 8). HGF-X7, in particular, showed the highest gene expressionlevel.

(2) Coexpression of HGF and dHGF

In order to examine coexpression of HGF and dHGF from hybrid HGF geneconstructs, total cellular RNAs were extracted from the transfected 293cells using the Trizol method (Trizol; Gibco BRL, USA) and subjected toRT-PCR to obtain cDNA. Then, using cDNA as a template DNA, PCRamplification was carried out using synthetic oligonucleotides of SEQ IDNOs: 17 and 18 as a primer pair. The PCR amplification mixture wasprepared by mixing 1 μl of the template DNA, 1 μl each of the primer (10pmol/μl), 10 μl of dNTP (10 mM), 3.5 unit of Taq polymerase (TAKARASHUZO, Japan) and 10 μl of enzyme buffer solution and adjusted to afinal volume of 100 a with distilled water. 30 cycles of PCRamplification was conducted, each cycle consisting of 1 min at 94° C., 1min at 55° C., and 90 sec at 72° C.

The amplified PCR products corresponded to the boundary region betweenexons 4 and 5 of HGF gene; HGF gene cDNA of 142 bp and dHGF gene cDNA of127 bp, respectively. With no splicing, the PCR product of at least 1 kbin length was amplified; and if alternative splicing occurred, HGF genecDNA of 142 bp and dHGF gene cDNA of 127 bp were simultaneouslysynthesized and amplified. The amplified PCR products were distinguishedby electrophoresis on a 12% polyacrylamide gel.

As shown in FIG. 9, while the bands of 142 bp and 127 bp were detectedin the lanes loading HGF gene cDNA and dHGF gene cDNA, respectively,both bands of 142 bp and 127 bp were detected in the lanes loadingHGF-X. The above results suggest that HGF and dHGF are simultaneouslyexpressed from hybrid HGF-X gene constructs of the present invention.

Example 3 Comparison of Expression Levels of HGF-X7, HGF Gene cDNA anddHGF Gene cDNA (In Vivo)

100 μg each of pCP-HGF-X7, pCP-cHGF and pCP-dHGF were injected into theinterior tibial muscle of the hind limb of mice with an insulin syringe.After 5 days, the mice were sacrificed and the muscles around theinjection spot were removed and smashed in a protein extraction buffer(25 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5% Na-deoxycholate, 2% NP-40,0.2% SDS) to separate total proteins. The amount of the total proteinswas measured with a DC protein analysis kit (Bio-Rad Laboratories, CA,USA) and the amount of expressed HGF was determined with an ELISA kit(R&D System) according to the manufacturer's instruction.

As can be seen from the result shown in FIG. 10, the amount of HGFexpressed from HGF-X7 is 250-fold higher than that from HGF gene cDNA ordHGF gene cDNA.

Together with the result of the experiment of Example 2 (in vivo), thisresult demonstrates that the expression efficiency of HGF-X gene is muchsuperior to those of HGF gene cDNA or dHGF gene cDNA.

Example 4 Gene Therapy Employing HGF-X7 in a Rabbit Ischemic Hind LimbModel

In order to examine whether HGF-X7 gene is effective in the treatment ofischemic hind limb disease, gene therapy was carried out on a rabbitischemic hind limb model as follows.

A rabbit ischemic hind limb model, which is a standard animal model forthe ischemic limb disease, was prepared by the method described byTakeshita et al., Journal of Clinical Investigation 93:662 (1994). Atthe day before operation (day 0), each of 30 white rabbits from NewZealand (male, from 3.8 to 4.2 kg) was intramuscularly injected with 5mg/kg of xylazine and, then, anesthetized by an intramuscular injectionof 50 mg/kg of ketamine. Subsequently, the left femoral region of therabbit was incised and all branches of the femoral artery were separatedand tied. The region from the proximal part to the branching point ofthe saphenous and popliteal arteries was incised to prepare the model.After the operation, 15 mg/kg/day of cefazolin was injectedintramuscularly for 5 days and 0.3 mg/day of morphin, for 10 days. 10days after the operation (day 10), angiography was carried out for theleft hind limb where the ischemia was induced, and the degree ofarteriogenesis was recorded as a basal level. The rabbits were randomlydivided into two groups and injected at four sites in the femoral musclewith 500 μg of plasmid pCP-HGF-X7(experimental group) or 500 μg ofplasmid pCP (control), respectively. 40 days after the operation (day40), angiography was carried out again for the left hind limb and thedegree of arteriogenesis at the arteriole level was determined andcompared to that of day 10.

As can be seen from the result in FIG. 11, the degree of angiogenesiswas significantly enhanced in the experimental group administered withpCP-HGF-X7 as compared with the pCP-administered control group.

This result demonstrates that HGF-X7 gene can be effectively used in thegene therapy of an ischemic disease.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

1-10. (canceled)
 11. A method of preventing or treating an ischemic limbdisease in a subject comprising administering to said subject apharmaceutical composition comprising: (a) a hybrid Hepatocyte GrowthFactor (HGF) construct comprising HGF exons 1-18 or degenerates thereofwhich do not alter the encoded amino acid sequence and an intron betweenexons 4 and 5; and (b) a carrier.
 12. The method of claim 11, whereinsaid intron is an inherent intron.
 13. The method of claim 12, whereinsaid hybrid HGF construct comprises a nucleotide sequence comprising SEQID NO:
 2. 14. The method of claim 11, wherein said hybrid HGF constructcomprises a nucleotide sequence not less than 80% identical to SEQ IDNO:
 2. 15. The method of claim 14, wherein said nucleotide sequence isnot less than 90% identical to SEQ ID NO:
 2. 16. The method of claim 15,wherein said nucleotide sequence is not less than 95% identical to SEQID NO:
 2. 17. The method of claim 11, wherein said intron is a fragmentof an inherent intron.
 18. The method of claim 11, wherein the sequenceof said hybrid HGF construct is not less than 80% identical to SEQ IDNO:
 19. 19. The method of claim 18, wherein said nucleotide sequence isnot less than 90% identical to SEQ ID NO:
 19. 20. The method of claim19, wherein said nucleotide sequence is not less than 95% identical toSEQ ID NO:
 19. 21. The method of claim 20, wherein said hybrid HGFconstruct comprises SEQ ID NO:
 19. 22. The method of claim 11, whereinthe sequence of said hybrid HGF construct is not less than 80% identicalto SEQ ID NO:
 20. 23. The method of claim 22, wherein said nucleotidesequence is not less than 90% identical to SEQ ID NO:
 20. 24. The methodof claim 23, wherein said nucleotide sequence is not less than 95%identical to SEQ ID NO:
 20. 25. The method of claim 24, wherein saidhybrid HGF construct comprises SEQ ID NO:
 20. 26. The method of claim11, wherein the sequence of said hybrid HGF construct is not less than80% identical to SEQ ID NO:
 21. 27. The method of claim 26, wherein saidnucleotide sequence is not less than 90% identical to SEQ ID NO:
 21. 28.The method of claim 27, wherein said nucleotide sequence is not lessthan 95% identical to SEQ ID NO:
 21. 29. The method of claim 28, whereinsaid hybrid HGF construct comprises SEQ ID NO:
 21. 30. The method ofclaim 11, wherein said hybrid HGF construct is comprised in a vector.31. The method of claim 30, wherein said vector further comprises one ormore sequences for regulating expression, a self-replication sequence,or a secretory signal.
 32. The method of claim 30, wherein said vectorcomprising said HGF construct is selected from the group consisting of:pCK-HGF-X1, pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X4, pCK-HGF-X5, pCK-HGF-X6,pCK-HGF-X7, pCK-HGF-X8, pCP-HGF-X1, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X4,pCP-HGF-X5, pCP-HGF-X6, pCP-HGF-X7 and pCP-HGF-X8.
 33. The method ofclaim 11, wherein said pharmaceutical composition is administered bydirect injection.
 34. A pharmaceutical composition for preventing ortreating an ischemic limb disease, which comprises (a) a hybridHepatocyte Growth Factor (HGF) construct comprising HGF exons 1-18 ordegenerates thereof which do not alter the encoded amino acid sequenceand an intron between exons 4 and 5; and (b) a carrier.
 35. A method ofpreventing or treating an ischemic limb disease in a subject comprisingadministering to said subject a pharmaceutical composition comprising:(a) a hybrid Hepatocyte Growth Factor (HGF) construct comprising apolynucleotide having a nucleic acid sequence not less than 90%identical to a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the polynucleotide havingsaid nucleic acid sequence encodes two heterotypes of human HGF; and (b)a carrier.